Name: identification of plasmodesmal localization sequences in proteins in planta
Uploaded: 2017-08-15T12:30:00
Description: Watch this Scientific Journal Video about Identification of Plasmodesmal Localization Sequences in Proteins In Planta at JoVE.com

For the lack of space, we cited mostly review articles, and we apologize to our colleagues whose original work was not cited. The work in the V.C. laboratory is supported by grants from NIH, NSF, USDA/NIFA, BARD, and BSF to V.C., and the S.G.L. laboratory is supported by NIH and funds from the Departments of Plant Pathology and Plant-Microbe Biology to S.G.L.

1. Plant Material
<ol>
	<li>Choice of plant species</li>
	<li>Use the plant species native to the protein of interest, i.e., one which encodes this protein for endogenous proteins or which represents the natural host for the pathogen for viral proteins. In addition, the selected plant species must be amenable to the chosen method of transient genetic transformation. 
	NOTE: The studies routinely employ Nicotiana benthamiana, which represents a good host for TMV and is transformed efficiently by t...

This protocol has four core constituents: the concept of identifying a sequence that is both necessary and sufficient for targeting to Pd, systematic division of the protein of interest into fragments that are progressively reduced in length, fusing the tested fragments to an autofluorescent protein that serves both as tag and as macromolecular cargo, and functional assay for Pd targeting in living plant tissues following transient expression of the tested fusion proteins. Note that Agrobacterium-mediated transient expre...

Plasmodesmata (Pd) function as conduits for intercellular transport of key regulators of plant development and morphogenesis, ranging from transcription factors to mRNA and small RNA molecules. Furthermore, this macromolecular transport capacity of Pd is utilized by most plant viruses for their intercellular spread during infection; to move through Pd, plant viruses have evolved specialized proteins, termed movement proteins (MPs), that specifically target to Pd1,2,3,4,5,6,7. Molecular pathways of Pd transport most likely are intimately interconnected with the specific sequences that target the transported proteins into these pathways. Thus, identification of these Pd localization signals (PLSs) may be diagnostic of the corresponding Pd transport pathway. This is by analogy of Pd transport8, for example, to different nuclear import pathways, which can be specific for different nuclear localization signal (NLS) sequences9,10. Conceptually, both NLSs and PLSs represent non-cleavable subcellular targeting sequences that are necessary and sufficient for targeting. However, unlike NLSs11, the sequence information about PLSs is severely limited. Specifically, only four protein sequences involved in Pd targeting have been reported, with all of them derived from endogenous plant proteins. The first one is represented by a homeobox domain of KN112 &#8211; a transcription factor that moves from inner cell layers to epidermis of the plant leaf13 &#8211; and its KNOX homologs14. The second one also is from a transcription factor, Dof, which contains a putative PLS described as the intercellular trafficking (IT) motif15. The third sequence is from the PDLP1 plasmodesmata-resident type 1 membrane protein, and it is represented by a transmembrane domain16. Finally, the fourth Pd targeting sequence was recently reported for glycosylphosphatidylinositol (GPI)-anchored proteins and it is represented by the glycosylphosphatidylinositol (GPI) modification signal17.
Interestingly, until very recently, no PLSs have been reported for viral MPs. Previous studies indicated the presence of putative PLS sequences in plant viral MPs18,19, but no true PLS, i.e., a minimal amino acid sequence both necessary and sufficient for Pd targeting of an unrelated cargo molecule (e.g., CFP) has been identified in a viral MP. Yet one of these proteins, MP of the Tobacco mosaic virus (TMV), was the first for which Pd localization and transport have been demonstrated20.
To address this gap, we developed an experimental strategy to identify TMV MP PLS. This strategy was based on three concepts. (i) We defined PLS as a minimal amino acid sequence that is both necessary and sufficient for protein targeting to Pd21. (ii) Because TMV MP first targets Pd and then translocates through these channels22, we aimed at uncoupling these two activities and identifying the bona fide PLS, which functions only for Pd targeting, and not for the subsequent transport. (iii) We analyzed the identified PLS for amino acid residues important for its Pd targeting activity, whether structurally or functionally. Using this approach, we delineated a 50-amino acid residue sequence at the amino-terminus of TMV MP that acts as bona fide PLS. This was done by producing a series of TMV MP fragments that saturated the entire length of the protein, tagging their carboxyl-termini with CFP and transiently expressing them in plant tissues. Pd localization of each of the tested fragments was determined by coexpressing them with a Pd marker protein, PDCB1 (Pd callose binding protein 1)23. The smallest fragment that still localized to Pd, but did not traverse Pd, was considered to represent PLS. Finally, the PLS was alanine-scanned to determine the key amino acid residues required for its structure and/or function.
Whereas here we illustrate this approach by describing identification of TMV MP PLS, it may be employed to discover PLSs in any other Pd-targeted proteins, whether encoded by plant pathogens or by the plants themselves; this is because our method does not take advantage of any unique features of viral MPs with regards to their ability to target to Pd.

<table border="0" cellpadding="0" cellspacing="0" width="1086">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">Confocal laser scanning microscope (CLSM)</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:268px;">LSM5</td>
			<td style="width:349px;">Any CLSM with similar capabilities is appropriate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zen software for confocal microscope imaging</td>
			<td>Zeiss</td>
			<td>2009 version</td>
			<td>The software should be compatible with the CLSM used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quickchange II site-directed mutagenesis kit&#160;</td>
			<td>Agilent</td>
			<td>200523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetosyringone</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td>D134406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td>69892</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringes without needles</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2</td>
			<td>FisherScientific</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectinomycin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S4014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rifampicin</td>
			<td>Sigma-Aldrich</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of plasmodesmal localization sequences in proteins in planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

The representative data, which faithfully illustrate the results expected from the described protocols and identify the TMV MP PLS, are adapted from Yuan et al.21. Figure 1A first summarizes major constructs expressing the full-length TMV MP (1-268), TMV MP PLS (comprising the first 50 amino acid residues of the protein, 1-50), and its alanine scanning V4A derivatives fused to CFP (generated as described in Steps 2.2, 5.2 and 6) whereas <s...

Watch this Scientific Journal Video about Identification of Plasmodesmal Localization Sequences in Proteins In Planta at JoVE.com

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

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